NMR Conformational and Dynamic Consequences of a Gly to Ser Substitution in an Osteogenesis Imperfecta Collagen Model Peptide

Abstract

Close packing of three chains in a standard collagen triple helix requires Gly as every third residue. Missense mutations replacing one Gly by a larger residue in the tripeptide repeating sequence in type I collagen are common molecular causes of osteogenesis imperfecta. The structural and dynamic consequences of such mutations are addressed here by NMR studies on a peptide with a Gly-to-Ser substitution within an ␣1(I) sequence. Distances derived from nuclear Overhauser effects indicate that the three Ser residues are still packed in the center of the triple helix and that the standard 1-residue stagger is maintained. NMR dynamics using H-exchange and temperature-dependent amide chemical shifts indicate a greater disruption of hydrogen bonding and/or increased conformational flexibility C-terminal to the Ser site when compared with N terminal. This is consistent with recent suggestions relating clinical severity with an asymmetric effect of residues N-versus C-terminal to a mutation site. Dynamic studies also indicate that the relative position between a Gly in one chain and the mutation site in a neighboring staggered chain influences the disruption of the standard hydrogen-bonding pattern. The structural and dynamic alterations reported here may play a role in the etiology of osteogenesis imperfecta by affecting collagen secretion or interactions with other matrix molecules.Mutations in collagen result in a variety of connective tissue diseases (1, 2), with the clinical phenotype depending on the location and function of the collagen type. For instance, mutations in type I collagen, the major collagen in bone, lead to a bone disorder, osteogenesis imperfecta (OI), 3 whereas mutations in type III collagen, which is present in high amounts in blood vessels, lead to aortic rupture in Ehlers-Danlos syndrome type IV (1, 2). All collagens have a triple helix motif composed of three polyproline II-like chains that are staggered by 1 residue and supercoiled about a common axis. The smallest residue Gly is typically present as every 3rd residue in each chain because of the tight packing of the chains, which generates the characteristic (Gly-Xaa-Yaa) n repeating sequence. The Gly residues are all buried in the center, and the structure is stabilized by interchain N-H (Gly) . . . CϭO (Xaa) hydrogen bonds (3-5). The most common type of mutation leading to collagen disorders is a missense mutation that replaces 1 Gly in the repeating sequence by a larger residue.The best characterized collagen disease is OI, or brittle bone disease, which is distinguished by fragile bones due to mutations in type I collagen (2, 6). More than 400 Gly substitution missense mutations in the ␣1(I) and ␣2(I) chains of type I collagen have been reported to lead to OI (7). The severity of the disease varies widely from mild cases with multiple fractures to perinatal lethal cases (2, 6, 7). A single base change in a Gly codon can lead to one of 8 residues (Ser, Ala, Cys, Val, Arg, Asp, Glu, Trp) or a missense mutation. The smallest residue Ala is...

Osteogenesis Imperfecta Missense Mutations in Collagen: Structural Consequences of a Glycine to Alanine Replacement at a Highly Charged Site

“…Despite the perturbations in hydrogen bonding, J-coupling values indicate the dihedral angles for the Ala16 residues did not vary significantly from those expected for a standard triple-helix. A previous study on a Gly to Ala substitution within a (Pro-Hyp-Gly) 10 peptide showed one of the three Ala residues had a J-coupling of 8.6 Hz, which is not compatible with a triple-helix structure [18], while all three Ala seem to be able to participate in a triple-helix structure in the current KAD context. Both Ala and Gly were found to be rigid on picoseconds time scale, suggesting any perturbations are occurring on a much longer time scale.…”

“…A value greater than −4.6 ppb/°C supports the presence of a hydrogen bond [23, 18]. The monomer resonances of Gly7 and Gly16 in peptide T1-655 and T1-655[G16A] at pH 7 show much more negative values than this cut-off value, indicating the absence of hydrogen-bonding (Figure 5B).…”

“…In addition, the angular distortion puts the two helical ends out-of- register. NMR studies on this peptide indicate that the Ala residues at the mutation site are non-equivalent, and that the amide of only one of the three Ala residues is capable of form a good hydrogen bond [18]. …”

Local Conformation and Dynamics of Isoleucine in the Collagenase Cleavage Site Provide a Recognition Signal for Matrix Metalloproteinases*

“…Residues in the triple helical conformation typically contain phi angles from Ϫ55 to Ϫ75 degrees and have a corresponding J coupling value of 4 -6 Hz (36,39,60 Dynamics of the Model Peptides-Hydrogen exchange experiments were performed on the five model peptides at all the labeled positions to explore the protection of amide protons from solvent (supplemental Table S1). Residue-specific local stabilities were calculated from the protection factors as described under "Experimental Procedures" (Fig.…”

“…NMR studies on peptide models of collagen are powerful approaches for probing the conformation and dynamics of individual chains of the triple helix directly at the specific residue level (35)(36)(37)(38)(39). Here we use NMR to characterize the differences in conformation and dynamics at the natural GlyϳIle-Ala cleavage site versus potential cleavage sites.…”